Background: Genome-wide association studies (GWAS) have identified numerous loci for complex diseases. This success has been most notable for the inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC), where ~100 genetic risk factors have been discovered, some that are phenotype-specific and others that are common to more than one disease. But, how do disease-associated genes and their corresponding alleles exert their influence on the biology of health and disease? The genetic data indicates that IBD can emerge from the perturbations of distinct signal transduction pathways, resulting from the dysregulation of disease-causative genes. Hypothesis and specific aims: Although there has been tremendous success for identifying IBD genetic risk loci using GWAS, this approach primarily leads to the identification of common variants, most of which have modest genetic effect. We postulate that rare variants within these loci might be responsible for the effects observed. To tackle this issue, we propose to perform targeted re-sequencing of validated IBD loci to identify the causal (rare) variation. With the advent of next-generation sequencing technologies and access to well-phenotyped cohorts, we are now able to use this innovative approach, which is complementary to our previous work using GWAS, and sequence UC-associated loci to identify rare variants of greater genetic effect. These rarer, more penetrant, alleles are expected to have a more significant biological impact thus increasing the feasibility and relevance of performing allele-specific functional studies as we propose herein. Intestinal inflammation is a complex process involving an interplay genetic predisposition and environmental factors and can be the result of the dysregulation of different biological processes involving specific cell types. In an attempt to dissect this interplay between genetics and environmental factors and understand the complexity of UC pathogenesis, we will complement our genomics approach with a systematic approach to functionally characterize UC-associated genes in conjunction with different environmental stresses. Given the diverse expertise of our teams, we will combine several functional approaches, including gene-expression analysis, shRNA and allele-specific rescue experiments in different cell types to place the genes and their variants under study in functional pathways in the relevant cell type and environmental context. We anticipate that these studies will point to a subset of key pathways that are perturbed in UC to define disease-mechanism clusters and will provide a more complete picture of the complex biological networks involved in UC. Our specific aims are: Aim 1) To identify risk and protective alleles within UC-associated regions identified by GWAS We want to (1A) identify novel uncommon and rare variants by deep re-sequencing in samples from patients and controls and (1B) validate these novel variants by genotyping large cohorts. Aim 2) To determine the function of UC-candidate genes We propose to take a systematic stepwise approach to place UC-associated genes within biological pathways. We will: (2A) Determine the RNA and protein expression of confirmed UC genes in primary cells and colon biopsies from UC patients and control individuals; (2B) Perform targeted over- expression and RNA interference (RNAi) screens using cell-based functional assays (2C) Determine the biological impact of selected genes (and their variants) at a cell-specific context. Impact: Understanding the mechanisms through which genetic variation contributes to UC, although challenging, offers exciting and promising advances in biomedicine, both by providing new insights into the genetic and molecular basis of UC pathophysiology and by helping to improve diagnosis and therapy. The investigation of causal genes (and their variants) linked to UC combined with our high-throughput and cell-type specific functional analysis will provide a mechanistic understanding of the role of genetic factors in UC. The identification of causal genetic variants linked to UC and their functional implications could provide new guidelines for drug dose adjustment, for changes in treatment modalities and for prevention strategies.